This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Microfocus X-ray Data Collection with Diffraction-Capable Microfluidic Chips and Microcrystals of Large Molecular Assemblies We have recently developed microfluidic crystallization chips designed for in-situ diffraction data collection. These chips have been designed for low-volume crystallization experiments (10nl sample per sample chamber) and to minimize x-ray background scatter from the chip material. Sections can be removed from chips, cryoprotectants can be added, and cryogenic data collection is possible. Crystals grown in these chips are typically small (40x40x40 [unreadable]m) and data collection from them will benefit from the use of microfocus X-ray sources to further reduce background X-ray scatter. This subproject aims to demonstrate the utility of the NE-CAT microfocus X-ray beamline for data collection from microfluidic chips and determine structures of samples of broad biological interest including DNA replication complexes, membrane proteins and bacterial virulence factors. We also have been studying large macromolecular assemblies that remodel and replicate DNA. Many of these systems assume multiple conformational states during their catalytic cycle, making them challenging structural targets, and prone to forming microcrystals despite extensive optimization efforts.